The complement system is the first line of immunological defense against foreign pathogens. Its activation through the classical, alternative or lectin pathways leads to the generation of anaphylatoxic peptides C3a and C5a and formation of the C5b-9 membrane attack complex. Complement component C3 plays a central role in activation of all three pathways. Activation of C3 by complement pathway C3 convertases and its subsequent attachment to target surface leads to assembly of the membrane attack complex and ultimately to damage or lysis of the target cells. C3 is unique in that it possesses a rich architecture that provides a multiplicity of diverse ligand binding sites that are important in immune surveillance and immune response pathways.
Inappropriate activation of complement may lead to host cell damage. Complement is implicated in several disease states, including various autoimmune diseases, and has been found to contribute to other clinical conditions such as adult respiratory syndrome, heart attack, rejection following xenotransplantation and burn injuries. Complement-mediated tissue injury has also been found to result from bioincompatibility situations such as those encountered in patients undergoing dialysis or cardiopulmonary bypass.
Complement-mediated tissue injuries are directly mediated by the membrane attack complex, and indirectly by the generation of C3a and C5a. These peptides induce damage through their effects on neutrophils and mast cells. In vivo, regulation of complement at the C3 and C5 activation steps is provided by both plasma and membrane proteins. The plasma protein inhibitors are factor H and C4-binding protein, and the regulatory membrane proteins located on cell surfaces are complement receptors 1 (CR1), decay-accelerating factor (DAF), and membrane cofactor protein (MCP). These proteins inhibit the C3 and C5 convertases (multi-subunit proteases), by promoting dissociation of the multisubunit complexes and/or by inactivating the complexes through proteolysis (catalyzed by factor I). Several pharmacological agents that regulate or modulate complement activity have been identified by in vitro assay, but most have been shown in vivo to be of low activity or toxic.
To date, there are no inhibitors of complement activation used in the clinic, though certain candidates for clinical use exist, specifically, a recombinant form of complement receptor 1 known as soluble complement receptor 1 (sCR1) and a humanized monoclonal anti-C5 antibody (5G1.1-scFv). Both of these substances have been shown to suppress complement activation in in vivo animal models (Kalli et al., Springer Semin. Immunopathol. 15: 417-431, 1994; Wang et al., Proc. Natl. Acad. Sci. USA 93: 8563-8568, 1996). However, each substance possesses the disadvantage of being large molecular weight proteins (240 kDa and 26,000 kDa, respectively) that are difficult to manufacture and must be administered by infusion. Accordingly, recent research has emphasized the development of smaller active agents that are easier to deliver, more stable and less costly to manufacture.
U.S. Pat. No. 6,319,897 to Lambris et al. describes the use of a phage-displayed combinatorial random peptide library to identify a 27-residue peptide that binds to C3 and inhibits complement activation. This peptide was truncated to a 13-residue cyclic segment that maintained complete activity, which is referred to in the art as Compstatin. Compstatin inhibits the cleavage of C3 to C3a and C3b by C3 convertase. Compstatin has been tested in a series of in vitro, in vivo, ex vivo, and in vivo/ex vivo interface experiments, and has been demonstrated to: (1) inhibit complement activation in human serum (Sahu et al., J. Immunol. 157: 884-891, 1996); (2) inhibit heparin/protamine-induced complement activation in primates without significant side effects (Soulika et al., Clin. Immunol. 96: 212-221, 2000); (3) prolong the lifetime of a porcine-to-human xenograft perfused with human blood (Fiane et al., Transplant. Proc. 31: 934-935, 1999a; Fiane et al., Xenotransplantation 6: 52-65, 1999b; Fiane et al., Transplant. Proc. 32: 899-900, 2000); (4) inhibit complement activation in models of cardio-pulmonary bypass, plasmapheresis, and dialysis extra-corporeal circuits (Nilsson et al., Blood 92: 1661-1667, 1998); and (5) possess low toxicity (Furlong et al., Immunopharmacology 48: 199-212, 2000).
Compstatin is a peptide comprising the sequence ICVVQDWGIEHRCT-NH2 (SEQ ID NO:1), where Cys2 and Cys12 form a disulfide bridge. Its three-dimensional structure was determined using homonuclear 2D NMR spectroscopy in combination with two separate experimentally restrained computational methodologies. The first methodology involved distance geometry, molecular dynamics, and simulated annealing (Morikis et al., Protein Science 7: 619-627, 1998) and the second methodology involved global optimization (Klepeis et al., J. Computational Chemistry, 20: 1344-1370, 1999). The structure of Compstatin revealed a molecular surface that comprises of a polar patch and a non-polar patch. The polar part includes a Type I β-turn and the non-polar patch includes the disulfide bridge. In addition, a series of analogs with alanine replacements (an alanine scan) was synthesized and tested for activity, revealing that the four residues of the β-turn and the disulfide bridge with the surrounding hydrophobic cluster are essential for inhibitory activity (Morikis et al., 1998, supra).
Using a complement activity assay comprising measuring alternative pathway-mediated erythrocyte lysis, the IC50 of Compstatin has been measured as 12 μM. Certain of the analogs previously tested have demonstrated activity equivalent to, or slightly greater than, Compstatin. The development of Compstatin analogs or mimetics with greater activity would constitute a significant advance in the art.